Manipulating matter at the nanometer (nm) scale is important for many electronic, chemical and biological advances (See Li et al., “Ion beam sculpting at nanometer length scales”, Nature, 412: 166–169, 2001). A number of sequencing techniques have been proposed at the micrometer and nanometer scale in response to the human genome project. These techniques have been largely developed to help characterize and understand expression of genes in vivo. A popular technique uses micro arrays and hybridization of cDNA to determine the presence or absence of a particular target gene. A target gene and probe are exposed in solution and bind if relative hybridization sequences match. Hybridization is indicative of the presence of the sequence or target gene. A dye may be employed with the target or probe to then determine existence and efficiency of hybridizations. The technique has been extended for use in determining the presence of single nucleotide polymorphism (SNP'S) in target cDNA. Micro arrays provide the promise of being able to accurately and concurrently screen for a variety of diseases in a particular patient. Theoretically, a patient should be able to enter a hospital, have blood taken, DNA extracted and genes sequenced. The sequencing methods provide for a genetic blue print of the individual. This provides patient specific information to doctors regarding susceptibility towards disease or existence of genetic abnormalities. A few major drawbacks of the micro array technique concern difficulty in manufacturing as well as the long time for effective hybridizations between probe and target (generally overnight to maintain high specificity). In addition, the large amounts of genomic DNA in a patient would require an inordinate amount of time and work. Therefore, new techniques are now being explored to more quickly sequence biopolymers. Systems that are on the nanoscale are both effective on resources (limited materials), but also may more closely mimic the processes already present in living cells (i.e. translocation processes). Therefore, nanopore technology has become a fundamental field area of interest to molecular biologists and biochemists alike.
It has been demonstrated that a voltage gradient can drive single-stranded biopolymers through a transmembrane channel, or nanopore (See Kasianowicz et al., “Characterization of individual polynucleotide molecules using a membrane channel”, Proc. Natl. Acad. Sci. USA, 93: 13770–13773, 1996). During the translocation process, the extended biopolymer molecule will block a substantial portion of the otherwise open nanopore channel. This blockage leads to a decrease in the ionic current flow of the buffer solution through the nanopore during the biopolymer translocation. The passage of a single biopolymer can be monitored by recording the translocation duration and the blockage current, yielding plots with predictable stochastic sensing patterns. From the uniformly controlled translocation conditions, the lengths of the individual biopolymers can be determined from the translocation time. Furthermore, the differing physical and chemical properties of the individual bases of the biopolymer strand can in principle generate a measurable and reproducible modulation of the blockage current that allows an identification of the specific base sequence of the translocating biopolymer.
Another method for detecting a biopolymer translocating through a nanopore has been proposed. This technique is based upon quantum mechanical tunneling currents through the portion of the translocating strand as it passes between a pair of electrodes. Measuring the magnitude of the tunneling current would be an electronic method of detecting the presence of a translocating biopolymer, and if the conditions were adequately controlled and the measurements sufficiently sensitive, the sequence of constituent bases could also be determined. One of the primary motivations for this approach is that typical tunneling currents in scanning tunneling microscopes are on the order of 1–10 nanoamps, which is two to three orders of magnitude larger than the ionic currents observed during polymer translocation of 2 nanometer nanopores, as described above (See Kasianowicz et al., “Characterization of individual polynucleotide molecules using a membrane channel”, Proc. Natl. Acad. Sci. USA, 93: 13770–13773, 1996).
Both of the techniques described above have major implementation challenges for detecting biopolymer translocation, characterizing the length of a stand, and ultimately performing sequencing of the constituent bases of the biopolymer. One of the primary difficulties is that the biopolymer is not constrained to pass through the center of the nanopore. Thus, there is an intrinsic variability between different translocation events, as well as potential variability during a single translocation as the possibility of lateral movement within the nanopore is assumed. The effects of this lateral displacement can be manifested in a number of ways for the two detection schemes described above.
For the first detection scheme that consists of measuring the magnitude of the reduced ionic current flow during translocation, lateral displacement of the translocating biopolymer can have two significant effects. First, if the biopolymer is moved away from the center of the nanopore, interactions with the walls of the nanopore itself would cause additional drag, causing the speed of the translocation to decrease. This variability would cause the measurement of the biopolymer length determined from the calibrated translocation time to be in error. In fact, it is not inconceivable that the translocating biopolymer could move far enough off the nanopore center that it could actually bind intermittently along the walls of the pore channel, either through molecular interactions or purely conformational binding of the biopolymer strand. The second significant effect that a lateral displacement of the translocating molecule would have is the potential change in the ionic blockage current. It is well known that the shape of an aperture can have significant effects on hydrodynamic flow. It is also self-evident that these effects become even more significant in the molecular flow regime, where the molecular size is on the order of the aperture. For this reason, it is expected that lateral displacement within the nanopore of the translocating biopolymer will cause significant variability in the magnitude of the measured ionic blockage current, making more difficult the job of differentiating the various bases by their blocking efficiencies, as described above.
For the second detection scheme, which consists of measuring quantum mechanical tunneling currents through the portion of the translocating biopolymer as it passes between a pair of electrodes, lateral displacement of the translocating strand can have two significant effects. As described for the first detection scheme, if the biopolymer is moved away from the center of the nanopore, interactions with the walls of the nanopore itself would cause additional drag, causing the speed of the translocation to decrease. This variability would cause the measurement of the biopolymer length determined from the calibrated translocation time to be in error. Secondly, it is well known that the tunneling current has an exponential dependence upon the height and width of the quantum mechanical potential barrier to the tunneling process. This dependence implies an extreme sensitivity to the precise location in the pore of the translocating molecule. Both steric attributes and physical proximity to the tunneling electrode could cause changes in magnitude of the tunneling current which would be far in excess of the innate differences expected between base-types under ideal conditions. For this reason, it is expected that lateral displacement within the nanopore of the translocating biopolymer will cause significant variability in the magnitude of the measured tunneling current, making more difficult the job of differentiating the various bases by their tunneling characteristics, as described above.
For all these reasons, variability of the lateral position of a biopolymer translocating a nanopore can cause significant problems, regardless of the detection scheme. A method of controlling the dynamics of the translocation process would provide many advantages over the present situation. These and other problems with the prior art processes and designs are obviated by the present invention. The references cited in this application infra and supra, are hereby incorporated in this application by reference. However, cited references or art are not admitted to be prior art to this application.